The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants

Abstract

<i>Listeria monocytogenes</i> is an opportunistic intracellular pathogen commonly associated with serious infections and multiple food-borne outbreaks. In this study, we investigated the influence of atmospheric cold plasma (80 kV, 50 Hz) on <i>L. monocytogenes</i> (EGD-e) and its knockout mutants of <i>sigB</i>, <i>rsbR</i>, <i>prfA</i>, <i>gadD</i>, and <i>lmo0799</i> genes at different treatment time intervals. Further, to ascertain if sub-lethal environmental stress conditions could influence <i>L. monocytogenes</i> survival and growth responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants were similarly affected after 1 min exposure to ACP (<i>p</i> > 0.05), with a difference in response noted only after 3 min of treatment. While all <i>L. monocytogenes</i> strains exposed to acid/cold stress were hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of treatment (<i>p</i> < 0.05). The results indicate <i>sigB</i> and <i>prfA</i> are important for general stress resistance and biofilm, respectively, loss of these two genes significantly reduced bacterial resistance to ACP treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress associated genes. <i>SigB</i> showed the highest gene expression, increasing by 15.60 fold, followed by <i>gadD2</i> (7.19) and <i>lmo0799</i> (8.6) after 1 min exposure. Overall, an increase in gene expression was seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced the gene expression and some cases down-regulated <i>prfA</i> and <i>gadD3</i> gene expression. By comparing the response of mutants under ACP exposure to key processing parameters, the experimental results presented here provide a baseline for understanding the bacterial genetic response and resistance to cold plasma stress and offers promising insights for optimizing ACP applications.